Interpretive Summary: The large-scale industrial production of cellulosic ethanol has been impeded by the high abundance of lignin in woody biomass. Current pretreatment methods to disrupt the lignin barrier are often costly, inefficient, and produce by-products that inhibit downstream fermentation processes. Communities of microbes associated with wood-feeding insects represent excellent candidates to prospect for novel lignin degrading enzymes that could overcome the limitations of current pretreatment methods. Through next generation sequencing, we analyzed the woody-biomass degrading capacity of gut microbes harbored by the Asian longhorned beetle, a wood-boring pest that has previously been shown to degrade lignin. We used a large-scale comparative metagenomic approach to identify lignin-degrading candidates in other microbial communities associated with wood feeding insects and compiled a suite of genes that could be explored for lignin-degrading potential. This is the first study to characterize an insect gut community of microbes that can degrade lignin with potential applications for industrial biofuels.

Technical Abstract:
The Asian longhorned beetle (Anoplophora glabripennis) is an invasive, wood-boring pest that thrives in the heartwood of deciduous tree species. The biggest impediment faced by A. glabripennis as it feeds on woody tissue is lignin, a highly recalcitrant biopolymer that reduces access to sugars locked in cellulose and hemicellulose. We previously demonstrated that lignin is depolymerized in the beetle’s gut and that the gut harbors an assemblage of microbes that could make significant contributions to this process. Further, unlike other wood-feeding insects that maintain obligate, external affiliations with wood-degrading fungi that play key roles in pre-digesting lignocellulose, A. glabripennis lacks external symbionts and the major lignin degrading reactions occur in the gut. While lignin degrading mechanisms have been well characterized in pure cultures of white rot basidiomycetes, little is known about such processes in microbial communities associated with wood-feeding insects. The goal of this study was to discover genes in the A. glabripennis gut microbiome that could facilitate digestion, and the removal of lignin. To accomplish this goal, we taxonomically and functionally characterized the A. glabripennis gut microbiota through amplicon and shotgun metagenome sequencing and a large-scale comparison with the metagenomes in a variety of other herbivore-associated communities. This analysis distinguished the A. glabripennis gut metagenome from the gut communities of other herbivores, including previously sequenced termite hindgut metagenomes. The A. glabripennis gut metagenome clustered with a fungal gallery community associated with Sirex wood wasp, which also has lignin degrading potential. We identified candidate lignin degrading genes that include laccases, dye-decolorizing peroxidases, and ß- etherases found in association with most wood-feeding insect communities. Additionally, novel peroxidases and hydrogen-peroxide generating enzymes were found in high abundances within the A. glabripennis gut relative to other herbivore-associated communities. These communities could serve as reservoirs of novel enzymes to enhance industrial biofuels production.